Trypsin is a highly valuable protease that has many industrial and biomedical applications. The growing demand for non-animal sources of trypsin with special properties has driven the interest to clone and express this protease in microorganisms. Reports about expression of recombinant trypsins show wide differences in the degree of success owing to difficulties associated with its expression and autocatalytic properties of the protein. The yeast Pichia pastoris appears to be the microbial host with the greatest potential for the production of trypsin.
Trypsin is a serine protease of ˜25 kDa, secreted by acinar cells of pancreas as an inactive precursor—trypsinogen. An activation peptide of amino acids DDDDK precedes the mature trypsin in trypsinogen, which is cleaved by enterokinase, in the intestinal lumen. The activated trypsin will cleave the proteins at the carboxyl-terminal end of accessible Arginine (R) and Lysine (K) amino acid residues. Trypsin will not only digest any protein containing accessible R and K, but also acts on the accessible R and K of its own sequence and degrades itself (autocatalytic activity). Hence it is a very big challenge to produce recombinant trypsin in microbial or mammalian systems. Moreover expression levels are very low. To overcome this problem, the inventors of the present invention have fused 97 amino acid Rhizopus oryzae lipase signal sequence to Bovine Trypsinogen and expressed it in Pichia pastoris. The presence of prolipase sequence stabilizes the expression of trypsinogen and appeared to prevent activation in vivo.
Prolipase acts as an N-terminal extension of lipase, distinct from the signal sequence which is necessary for the transport of the protein into or through the membrane, or for its secretion into the extracellular medium. The 69 amino acid propeptide region of the Rhizopus oryzae lipase immediately follows the 26-amino acid signal sequence. Previous studies have shown that a mutation (C56 to S) in the prolipase region slows down the folding of lipase (Beer H. D., Wohlfahrt G., Schmid R. D., McCarthy J. E. G., Biochem. J. 319:351-359, 1996). The replacement of proregion of native bovine trypsinogen with prolipase region from Rhizopus oryzae lipase, surprisingly improved the stability and the yield of the recombinant bovine trypsinogen.
The known state of art fails to provide a generally applicable method of producing in a satisfactorily/optimally purifiable form of heterologous fusion polypeptides, expressed in a suitable host cell.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method devoid of the above limitation.
The modified novel Prolipase-Bovine trypsinogen fusion protein of the present invention, overcomes the above-discussed and numerous other disadvantages and deficiencies of the prior art.